PROJECT SUMMARY/ABSTRACT Cleavage of amyloid precursor protein (APP) by b-site APP cleaving enzyme-1 (BACE-1) is the rate- limiting step in production of Ab, whose deposition is the pathological hallmark of Alzheimer?s disease (AD). Despite a rapidly growing burden of health care for the aging United States, there is a fundamental gap in understanding how trafficking and mutations of APP influence neuronal function and contribute to AD pathogenesis. Continued existence of this gap represents a critical problem because, until it is filled, AD prevention and treatment based on molecular understanding of the disease progression remains inaccessible. Since neurons can grow axons that are up to a meter long, continuous imaging of APP trafficking and processing in live neurons at the single-molecule level requires extremely photostable fluorophores. Our lab has recently developed a new class of upconversion nanoparticles (UCNPs) that are immensely photostable over months. The overall objective of this project is to use our novel photostable UCNPs to perform single-molecule imaging of the trafficking and processing of APP in live human induced neurons (iNs) ? an excellent model system as many human diseases are not fully recapitulated in mouse neurons. The central hypothesis is that mutations of APP lead to impaired axonal transport and render APP more vulnerable for b-cleavage by BACE-1. This hypothesis has been formulated on the basis of previous work on culture mouse neurons and transgenic mouse models. The rationale for the proposed research is that ultralong-term single molecule imaging of WT and mutant APP in human iNs will reveal axonal transport defects caused by AD-associated mutations, providing important insights into their relationship to AD. Guided by strong preliminary data on the novel experimental platform, the hypothesis will be tested by pursuing the three specific aims: 1) Measure the trafficking dynamics of endocytosed APP in human iNs; 2) Determine how axonal transport is impaired by mutations of APP in human iNs; and 3) Visualize the association dynamics of APP and BACE-1 in human iNs. A battery of techniques including single-molecule imaging, nanotechnology, biochemistry and stem cell technology will be used to interrogate APP trafficking. The approach is innovative because it departs from the status quo by utilizing extremely photostable UCNPs to perform long-term single- molecule tracking, novel analysis for non-invasive determination of motor number, and the use of human induced neurons. The proposed research is significant because it is expected to characterize in depth the trafficking and association dynamics of APP and BACE-1 with unprecedented spatiotemporal resolution, as well as uncover the extent to which APP mutations impair axonal transport, thereby shedding light on future prevention and treatment of AD.